A network of genes, including cell cycle regulatory genes, proto-oncogenes, and tumor suppressor genes have emerged, which play major roles in normal physiological processes as well as in tumor progression (Grana and Reddy, Oncogene 11 (1995), 221-219; Hartwell and Kastan, Science 266 (1994), 1821-1828; Hoffman and Liebermann, Oncogene 9 (1995), 1807-1812; Sherr, Cell 79 (1994), 551-555). Oncogenes have first captured the lion's share of attention in the molecular and genetic studies on cell transformation. But it has now been realized that there is an equally important second side of the coin, presented by a distinct class of genes known variously as tumor suppressor genes (TSGs) or anti-oncogenes. Logic dictates that there must exist an equally elaborate array of growth-constraining elements in the cell's signaling circuitry that serve to counteract the growth-promoting proto-oncogenes (Fisher, Cell 78 (1994), 539-542; Karp and Broder, Nature Med. 1 (1995), 309-320; Liebermann et al., Oncogene 11 (1995), 119-210; Thompson, Science 267 (1995), 1456-1462). These tumor suppressor genes are of special interest since they may open up new possibilities for the treatment of cancers of various kinds and may help to better understand the molecular mechanisms responsible for the development of cancer.
The isolation of such suppressor genes has become feasible by progress in various fields with major contributions of molecular genetics and cell cycle analysis. Molecular genetics applied linkage studies to the isolation of TSGs, but the most fruitful strategies have evolved from the study of the genetic mechanisms employed by nascent tumor cells to discard their second, surviving copy of a tumor suppressor gene which results in homozygosity at the tumor suppressor locus. This event can often be traced by following the fate of anonymous DNA markers whose polymorphism allows detection of hetero- and homozygous states in these chromosomal regions. By this strategy the identification of the retinoblastoma gene product (Rb), the Wilms tumor suppressor gene (WT) and the von Hippel-Lindau tumor-suppressor gene has been possible. Most recently the cloning of the breast cancer susceptibility genes, BRCA1 and BRCA2 (Miki et al., Science 266 (1995), 66-71; Wooster et al., Nature 378 (1995), 789-792) has been accomplished by this approach.
Yet, the vast majority of human cancers, including breast cancer, develop spontaneously or under poorly defined criteria of genetic susceptibility preventing linkage studies to perform and indicating that epigenetic mechanisms appear to play the major role in the initiation and formation of tumors, which seem to develop in a multi-step process.
Further support for the concept of TSGs came up with the characterization and isolation of the regulatory components of the mammalian cell cycle. This progress has led to the identification of a new class of candidate tumor suppressor genes, the ubiquitously expressed cyclin-dependent kinase inhibitors (cdk), which negatively regulate cell cycle progression. Among the various forms described so far (p15, p16, p18, p21 and p27) the cdk p16 has been demonstrated to be mutated in-vivo in a spectrum of tumors examined (Marx, Science 264 (1994), 344-345; Kamb et al., Science 264 (1994), 436-440; Nobori et al., Nature 368 (1994), 753-756).
Another important example of a tumor suppressor gene is the p53 TSG, whose biological activity has been elucidated in-vitro through molecular and biochemical studies before it became identified as the genetic cause of the Li-Fraumeni syndrome. It is one of the most frequently mutated tumor suppressor genes in human tumors from various origins (Hollstein et al., Science 253 (1991), 49-53). This TSG encodes a transcription factor with two important functional properties contributing to its growth-suppression function: induction of apoptosis and cell cycle arrest (Vogelstein and Kinzler, Cell 70 (1992), 523-526; Oren, FASEB J. 6 (1992), 3169-3176; Perry, Curr. Opin. Genet. Dev. 3 (1993), 50-54; Bates and Vousden, Curr. Opin. Genet. Dev. 6 (1996), 12-19).
Although tumor suppressor genes have recently attracted a lot of attention due to the possibility that they may provide important targets in the treatment of cancer, only a limited number of TSGs could be identified and cloned. Thus, there still exists a need for the identification of further tumor suppressor genes in order to better understand the mechanisms of the development of diseases such as cancer and to be able to provide means for the treatment of further forms of tumorous diseases or for the improved treatment of tumorous diseases. One reason for the slow progress in cloning TSGs may be seen in the fact that there exists no method for the identification and isolation which can be easily carried out in-vitro and allows the rapid screening of a plurality of potential sequences for tumor suppressor activity.
Thus, the technical problem underlying the present invention is to provide further nucleic acid molecules coding for proteins displaying tumor suppressor activity as well as methods for their identification and isolation.